high throughput analysis of phthalates and parabens in ... · shaving cream. triclocarban is also...
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WAT E R S SO LU T IO NS
ACQUITY UPLC H-Class System
ACQUITY UPLC BEH Column
ACQUITY QDa Detector
Empower® 3 CDS Software
K E Y W O R D S
Phthalates, parabens, triclocarban,
consumer products, cosmetics,
personal care products
A P P L I C AT IO N B E N E F I T S
The ACQUITY® QDa® Detector linked to the
ACQUITY UPLC® H-Class System provides
improved confidence in the identification and
quantification of phthalates and parabens in
cosmetics and personal care products offering:
■■ Increased sample throughput and a reduction
of solvent usage due to reduced run times.
■■ Improved sensitivity, selectivity,
and robustness, compared with
existing methodologies.
■■ Cost effective, reliable mass confirmation.
IN T RO DU C T IO N
Phthalates are esters of phthalic acid that have extensively been used as
plasticizers to increase flexibility, transparency, durability, and longevity in
a wide variety of consumer and household products, such as children’s toys,
electronics, clothes, flooring, wallpaper, and paints. Phthalates are also used,
as plasticizers, solubilizers, or denaturants in cosmetics and personal care
products, such as perfumes, nail polishes, and hair sprays.
Parabens are esters of parahydroxybenzoic acid, which due to their low
volatility, high stability, antibacterial and antifungal properties, have
been used as preservatives in cosmetics, personal care, pharmaceutical,
food, and industrial products.
Triclocarban is an antibacterial and antifungal agent that is used in many cosmetic
and personal care products, including soap, toothpaste, deodorant, shampoo and
shaving cream. Triclocarban is also used in several consumer products including
kitchen cutting boards, shoes, towels, and clothing, as well as in medical
disinfectants and medical products. But there are several health concerns related
to the use of triclocarban, including potential hormone and endocrine disruption,
and also its potential to contribute to the development of antibiotic resistance.
Many phthalates are classified as hazardous because of their effects on the
reproductive system and their association with an increased risk of cancer. Parabens
are associated with allergenic contact dermatitis and rosecea. Studies1,2 have
also suggested parabens may be carcinogenic and possess estrogenic disrupting
activities. Due to these properties phthalates, parabens, and triclocarban are either
banned or restricted, as regulated by the Cosmetic Directive 1223/2009.3
In order to accommodate consumer demands for higher standards, many
manufacturers are developing, and labeling cosmetic and personal care
products ‘free from’ phthalates and parabens.
Previous example methodologies for the analysis of phthalates include
GC-MS,4 and HPLC-UV4; GC-FID,5 HPLC-UV,4,6 HPLC-MS,7 GC-MS,4 and capillary
electrophoresis6 for the analysis of parabens; and HPLC-MS8 for the analysis
of triclocarban.
High Throughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products Using UPLC with Mass Detection and Empower 3 SoftwareJane CooperWaters Corporation, Wilmslow, UK
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E X P E R IM E N TA L
Sample preparation
Cosmetic and personal care sample analysis ■■ Add 2.5 mL water and 2.5 mL methanol to 0.2 g sample.
■■ Vortex mixture for 2 minutes (1600 rpm).
■■ Further extract mixture in an ultrasonic bath for 30 minutes.
■■ Centrifuge approximately 1 mL of extract for 5 min
(10,000 rpm).
■■ Transfer centrifuge extract to LC vials for analysis.
LC conditionsLC system: ACQUITY UPLC H-Class
Runtime: 5.00 min
Column: ACQUITY UPLC BEH C18, 1.7 µm, 2.1 x 50 mm
Column temp.: 40 °C
Sample temp.: 10 °C
Mobile phase A: Water + 0.1% formic acid
Mobile phase B: Methanol + 0.1% formic acid
Flow rate: 0.6 mL/min
Injection volume: 5.0 µL
Mobile phase gradient is detailed in Table 1.
Time Flow rate (min) (mL/min) %A %B Curve
1 Initial 0.60 30 70 –
2 1.00 0.60 30 70 6
3 1.50 0.60 10 90 6
4 4.00 0.60 10 90 6
5 4.01 0.60 30 70 6
6 5.00 0.60 30 70 6
MS conditionsMS system: ACQUITY QDa
Ionization mode: ESI + and -
Capillary voltage: 0.8 kV
Probe temp.: 450 °C
Acquisition: Selected Ion Recording (SIR)
Cone voltage: 15 V
The list of compounds considered, including phthalates, parabens,
and triclocarban, along with their expected retention times are
detailed in Table 2. The empirical formulas and structures are
detailed in Tables 3 and 4.
ESI ionization mode (-/+)
SIR (m/z)
Retention time (minutes)
Diethyl phthalate + 223.1 0.37
Dipropyl phthalate + 251.1 0.58
Dibutyl phthalate + 279.2 1.12
Benzylbutyl phthalate + 313.4 1.07
Bis(2-ethylhexyl) phthalate
+ 391.3 2.92
Diisobutyl phthalate + 279.2 1.04
Di-n-pentyl phthalate + 307.2 2.10
Di-n-hexyl phthalate + 335.2 2.44
Dicyclohexyl phthalate + 331.2 2.09
Di-(2-methoxyethyl)-phthalate
+ 283.1 0.28
Di-n-octyl phthalate + 391.3 3.10
Methylparaben - 151.1 0.27
Ethylparaben - 165.0 0.30
Propylparaben - 179.0 0.35
Butylparaben - 193.1 0.44
4-Hydroxybenzoic acid - 137.0 0.24
Benzylparaben - 227.1 0.43
Triclocarban - 315.0 1.07Table 1. ACQUITY UPLC H-Class mobile phase gradient.
Table 2. Phthalates, parabens, and triclocarban; ionization mode, SIR m/z, and expected retention times.
Accessible and intuitive as an optical detector, the ACQUITY QDa Detector has been designed for chromatographers with ease of use
in mind. Mass detection can be used to achieve reliable analytical methods to unequivocally identify and quantify compounds such as
phthalates, parabens, and triclocarban, during both method development stages, and during routine regulatory analysis.
This application note considers the method development, sample extraction, and mass spectral analysis of parabens, phthalates,
and triclocarban using Waters® ACQUITY UPLC H-Class System, coupled to the ACQUITY QDa Detector.
High T hroughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products
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Table 3. Phthalates, associated CAS numbers, empirical formulas, and structures.
Table 4. Parabens and triclocarban, associated CAS numbers, empirical formulas, and structures.
Instrument control, data acquisition, and result processing
Empower 3 Software was used to control the
ACQUITY UPLC H-Class System and the
AQCUITY QDa Detector, as well as for
data acquisition and quantitation.
High T hroughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products
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R E SU LT S A N D D IS C U S S IO N
Figure 1. SIR chromatograms for phthalates, parabens, and triclocarban in a mixed 1.0 µg/mL calibration standard.
A fast, selective, and sensitive LC-MS method for the
detection of a selection of phthalates, parabens, and
triclocarban in cosmetic and personal care products
has been developed.
The ACQUITY QDa Detector’s SIR parameters were
optimized, considering both negative and positive
electrospray ionization modes, in order to ensure full
coverage of all the compounds being analyzed (as
detailed in Table 2.)
Method development was carried out using reversed
phase UPLC,® where different gradient conditions,
columns, and mobile phases were considered. The
objective was to separate the isomeric phthalate
compounds considered: di-n-octyl phthalate (DiNP),
and diisobutyl phthalate (DiBP); bis(2-ethylhexyl)
phthalate (DEHP), and di-n-octyl phthalate (DnOP)
– while maintaining sample throughput. This was
achieved by optimizing the mobile phases and
the gradient eluting conditions used. The final LC
conditions used are detailed in the methods section.
The method was established over the calibration
ranges of 0.01 µg/mL to 10 µg/mL for phthalates
and triclocarban, and 0.05 µg/mL to 25 µg/mL
for parabens, equivalent to 0.25 to 250 mg/Kg,
and 1.25 to 625 mg/Kg in the extracted samples
respectively. Good linearity was achieved for
all the compounds considered (R2 >0.99). SIR
chromatograms for phthalates, parabens, and
triclocarban in a mixed 1.0 µg/mL calibration
standard are shown in Figure 1.
The developed five-minute UPLC method, is more
than seven times faster than existing HPLC and GC
methods, with an excess of 90% less solvent usage
than existing HPLC methods.
High T hroughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products
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The SIR mass detection conditions detailed in
Table 2 were used after appropriate sample
preparation to screen for phthalates, parabens, and
triclocarban in cosmetic and personal care samples.
Cosmetic and personal care sample analysis
Samples were fortified at various levels with
selected phthalates and parabens, then prepared
for analysis as detailed in the experimental section.
Example SIR chromatograms achieved are shown
in Figure 2.
Figure 2. SIR chromatograms for selected phthalates and parabens in hair conditioner.
High T hroughput Analysis of Phthalates and Parabens in Cosmetics and Personal Care Products
Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com
Waters, ACQUITY, ACQUITY UPLC, QDa, UPLC, Empower, and The Science of What’s Possible are registered trademarks of Waters Corporation. All other trademarks are the property of their respective owners.
©2015 Waters Corporation. Produced in the U.S.A. October 2015 720005521EN AG-PDF
CO N C LU S IO NS■■ A fast, robust, and sensitive method was developed for the combined
analysis of phthalates, parabens, and triclocarban in cosmetic and
personal care samples.
■■ The ACQUITY QDa Detector provides cost effective reliable mass confirmation,
during both method development and routine analysis.
■■ Combining the ACQUITY UPLC H-Class System with the ACQUITY QDa
Detector offers accurate and reproducible quantification.
■■ Empower Chromatography Data Software provides confidence in data
management, data processing, and reporting.
■■ The developed 5-minute UPLC method is more than 7 times faster than
existing HPLC and GC methods, with an excess of 90% less solvent usage
than existing HPLC methods.
■■ The ACQUITY H-Class System, a quarternary system based on UPLC
Technology, offers the best in chromatographic resolution, and sensitivity.
References
1. Darbe P D. Environmental oestrogens, cosmetics and breast cancer. Best Practice & Research Clinical Endocrinology & Metabolism. 20(1): 121–143, 2006.
2. Golden R, Gandy J, Vollmer G. A review of the Endocrine activity of parabens and implications for potential risks to human health. Critical reviews in toxicology. 35(5): 435–458, 2005.
3. The European Parliament and the Council of the European Union. Regulations (EC) No 1223/2009 of The European Parliament and of the Council of 30 November 2009 on Cosmetic Products. Official Journal of the European Union. L 342: 59–209, 30th November 2009. [cited 2015 August 25]. Available from: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:342:0059:0209:en:PDF
4. Shen H Y, Jiant H L, Mao H L, et al. Simultaneous determination of seven phthalates and four parabens in cosmetic products using HPLC-DAD and GC-MS methods. J Sep Sci. 30: 48–54, 2007.
5. Farajzadeh M A, Djozan Dj, et al. Use of a capillary tube for collecting an extraction solvent lighter than water after dispersive liquid-liquid microextraction and its application in the determination of parabens in different samples by gas chromatography-flame ionization detection. Talanta. 81: 1360, 2010.
6. Labat L, Kummer E, Dallet P, Dubost JP. Comparison of high-performance liquid chromatography and capillary zone electrophoresis for the determination of parabens in a cosmetic product. J Pharm Biomed Anal. 23:763, 2000.
7. González-Mariño I, Quintana J B, Rodríguez, Cela R. Evaluation of the occurrence and biodegradation of parabens and halogenated by-products in wastewater by accurate-mass liquid chromatography-quadrupole-time- of-flight-mass spectrometry (LC-QTOF-MS). Water Research. 20: 6770–6780, 2011.
8. Wang Y, Li P, Liu Y, et al. Determination of Triclocarban, Triclosan and Methyl-Triclosan in Environmental Water by Silicon Dioxide/Polystyrene Composite Microspheres Solid-Phase Extraction Combined with HPLC-ESI-MS. Journal of Geoscience and Environment Protection. 1(2): 13–17. 2013.